Abl promotes cadherin-dependent adhesion and signaling in myoblasts

Abstract

Cell-cell contact promotes myogenic differentiation but the mechanisms that regulate this phenomenon are not well understood. Cdo (also known as Cdon), an Ig superfamily member, functions as a component of cell surface complexes to promote myogenic differentiation through activation of p38α/β MAP kinase. We recently showed that N-cadherin ligation activated p38α/β in a Cdo-dependent manner, whereas N-cadherin ligation-dependent activation of ERK MAP kinase was not affected by loss of Cdo. The non-receptor tyrosine kinase Abl associates with Cdo during myoblast differentiation and is necessary for full activition of p38α/β during this process. The Abl SH3 domain binds to a PxxP motif in the Cdo intracellular domain, and both these motifs are required for their promyogenic activity. Here we show that Abl is necessary for p38α/β activation initiated by N-cadherin ligation, but in contrast to Cdo, Abl is also required for N-cadherin-dependent ERK activation. Moreover, Abl is required for efficient cadherin-mediated myoblast aggregation via modulation of RhoA-ROCK signaling. Therefore, Abl regulates N-cadherin-mediated p38α/β activation by multiple mechanisms, more generally through regulation of cell-cell adhesion and specifically as a component of Cdo-containing complexes. The role of Cdo as a multifunctional coreceptor with roles in several pathways is also discussed.

Introduction

The development of skeletal muscle is a tightly regulated process, in which transcription factors such as myogenic regulatory factors (MRF) and myocyte enhance factor-2 (MEF2) play a crucial role in specification and differentiation of cells in the skeletal muscle lineage.^1,2 The activity of these transcription factors is promoted by the p38α/β mitogen-activated protein kinase (MAPK) signaling pathway, which is also crucial for skeletal muscle differentiation. p38α/β is activated during myogenic differentiation in vitro, and treatment of myoblasts with specific chemical inhibitors of p38α/β prevents differentiation.^3–5 p38α-deficient myoblasts show reduced recruitment of the transcriptional machinery to muscle loci and delayed cell cycle exit, resulting in impaired differentiation.⁶ Mice lacking p38α exhibit increased myoblast proliferation and delayed myofiber growth and maturation.⁶ p38α/β positively regulates myogenesis by direct phosphorylation of proteins that promote muscle-specific gene expression, including MEF2, the BAF60 subunit of the SWI/SNF chromatin remodeling complex, E47 and the RNA decay-promoting factor KSRP.^5,7–9

Environmental signals provided by cell-cell contacts are also involved in the regulation of myogenesis. Several classical cadherins (e.g., N-, R- and M-cadherin) are expressed in embryonic muscles during mouse development and have been implicated in regulation of myogenesis. N-cadherin promotes myoblast differentiation in the embryo and in vitro, although it is not essential for this process, perhaps due to redundancy with other cadherins expressed in skeletal muscle.^10–14 However, how N-cadherin ligation transduces signals to regulate myogenesis is not clear.

Cdo, a cell surface receptor of the Ig superfamily, is expressed on developing muscles and muscle precursor cells and positively regulates myogenesis. Mice lacking Cdo display delayed skeletal muscle development and Cdo^-/- myoblasts differentiate defectively in vitro.^15,16 Similarly, RNAi-mediated depletion of Cdo in C2C12 myoblasts reduces myogenic differentiation.^16–18 During myoblast differentiation, the intracellular region of Cdo binds to the p38α/β pathway scaffold protein JLP and, via JLP, p38α/β itself.¹⁶ The Cdo intracellular region also interacts with Bnip-2, a protein that binds the small GTPase, Cdc42. The Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38α/β activity and myoblast differentiation.¹⁸ A similar pathway has also been identified to regulate neuronal differentiation in vitro.¹⁹

N-cadherin Ligation Initiates Cdo-Dependent p38α/β MAPK Signaling in Skeletal Myoblasts

The observations that Cdo forms cis complexes with N-cadherin²⁰ and that cell-cell contact between myoblasts promotes p38α/β activity,^5,21 raised the possibility that cadherin-mediated adhesion regulates p38α/β activity through Cdo/Bnip-2/JLP complexes. Indeed, we recently reported that cadherin ligation activates p38α/β in a largely Cdo-, JLP- and Bnip-2-dependent manner. C2C12 myoblasts plated on N-cadherin ectodomain-coated substrates, but not poly-L-lysine (PLL)- or fibronectin-coated substrates, induce p38α/β phosphorylation in a manner sensitive to RNAi-mediated depletion of Cdo, JLP or Bnip-2.²² In contrast, activation of another MAP kinase, ERK, triggered by N-cadherin ligation is not significantly affected by depletion of Cdo, JLP or Bnip-2, indicating that these factors are necessary and specific for p38α/β activation induced by N-cadherin ligation.²² Furthermore, Cdo, JLP and Bnip-2 cluster at sites of N-cadherin ligation, with JLP and Bnip-2 doing so in a Cdo-dependent manner.²²

Abl is a ubiquitously expressed non-receptor protein tyrosine kinase involved in many signaling processes.²³ Abl shuttles between the nucleus and cytoplasm. Upon myoblast differentiation, Abl exits the nucleus and accumulates in the cytoplasm and cytoplasmic, but not nuclear, Abl promotes myogenic differentiation.²⁴ During myoblast differentiation, Abl binds to a PxxP motif in the cytoplasmic tail of Cdo via its SH3 domain, and both these motifs are necessary for their promyogenic activity.¹⁷ Abl also binds to JLP.¹⁷ Furthermore, Abl is necessary for full activation of p38α/β MAPK during myogenesis in vitro.¹⁷ Abl kinase activity is relatively low in growth medium but induced when cells reach confluence, and inhibition of cadherin-based adhesion by calcium chelation diminishes Abl kinase activity in differentiating myoblasts.¹⁷ Together, these results suggest Abl may also be involved in N-cadherin-initiated p38α/β activation.

Abl Promotes N-cadherin-Dependent Signaling by Regulation of Cell-Cell Adhesion

To test the notion that Abl is involved in p38α/β activation initiated by N-cadherin ligation, C2C12 cells were depleted of Cdo or Abl by RNAi (Fig. 1A), and the cells were plated at low density (without any cell-cell contact) on culture dishes coated with N-cadherin ectodomain-Fc fusion protein (Ncad-Fc); cells that expressed a control RNAi sequence were also analyzed. Alternatively, dishes coated with PLL were used as controls. Cells were then harvested after 2 hours. Cells plated on Ncad-Fc produced the phosphorylated (activated) form of p38α/β (pp38), whereas cells plated on PLL did not (Fig. 1B). Moreover, depletion of either Cdo or Abl diminished production of pp38 upon N-cadherin ligation to an extent that was roughly proportional to the extent of knockdown (Fig. 1A and B). Phosphorylation of another MAP kinase, ERK, was also induced by plating cells on Ncad-Fc, but not PLL. Surprisingly, while depletion of Cdo did not significantly affect ERK activation by N-cadherin ligation, ERK phosphorylation was diminished by the knockdown of Abl (Fig. 1B).

Figure 1.

Abl is involved in Ncad-induced p38α/β and ERK activation. (A) Western blots of C2C12 cells stably expressing siRNA against Cdo, Abl or a control siRNA (Con), were probed with antibodies against Abl, Cdo or Cadherin. (B) Western blot analysis of pp38, total p38α/β, pERK and total ERK2 in C2C12 cells expressing the indicated siRNA constructs and cultured for 2 hr on Ncad-Fc (Ncad) or PLL substrates.

While previous results demonstrated that Abl could specifically cooperate with Cdo and JLP to activate p38α/β,¹⁷ the observation that knockdown of Abl decreased activation of both p38α/β and ERK suggests that Abl may regulate N-cadherin-induced signaling by additional mechanisms. Abl has been implicated in the regulation of cytoskeletal processes, including formation of lamellipodia and filopodia and cell-cell adhesion.^25,26 Therefore, Abl may also regulate p38α/β activation indirectly through modulation of cadherin-mediated adhesion.

To determine whether Abl is involved in the regulation of cadherin-mediated myoblast adhesion, we took advantage of the hanging drop aggregation assay that has been widely used to study the cellular adhesive properties of cadherins.²⁷ A suspension of single cells is plated in 20 µl drops, which are allowed to hang from the lid of cell culture dishes and the formation of cell aggregates is monitored over time. The extent of cell aggregation is represented by the aggregation index (N_o – N_t)/N_o where N_t is the total particle number after the incubation time t, and N_o is the total particle number at the initiation of incubation.²⁷ Control C2C12 cells formed aggregates after 15 min with an aggregation index of ∼0.8. Aggregation was inhibited by preincubation of the cells with the calcium chelator EGTA, a characteristic property of cadherin-dependent adhesion (Fig. 2A). C2C12 cells depleted of Cdo did not show any significant defect in aggregation, consistent with the finding that only p38α/β, but not ERK, activation is diminished when such cells were plated on Ncad-Fc-coated culture dishes. However, RNAi-mediated depletion of Abl strongly decreased the efficiency of aggregation, dropping the aggregation index to ∼0.5 (Fig. 2B and D). After 30 min, Abl-depleted cells had aggregated but the aggregates formed were much smaller than those from control cells and cells depleted of Cdo (Fig. 2C and E). Loss of Abl did not alter the cadherin expression level (Fig. 1A). These data reveal that Abl is important for cadherin-mediated C2C12 myoblast adhesion.

Figure 2.

Effect of siRNA-mediated knockdown of Cdo and Abl on C2C12 cell aggregation. (A) C2C12 cells treated with or without EGTA (1.8 mM) for 8 hr were placed in 20 µl drops hanging from the lids of cell culture dishes. They were analyzed after 15 min. (B and C) C2C12 cells stably expressing siRNA against Cdo, Abl or a control siRNA (Con) were also subjected to the hanging drop assay and were analyzed after 15 min (B) or 30 min (C). C2C12 cells depleted of Abl by RNAi were cultured with or without 10 µM Y27632. (D) Aggregation after 15 min was quantified with an aggregation index ((N₀ − N_t)/N₀) as described in the text. Higher values represent a higher degree of aggregation. (E) Aggregation after 30 min was quantified measuring the average area of cell clusters by imageJ *p < 0.01 by Student's t-test. (Scale bars, 1 mm).

Abl Regulates Intercellular Adhesion through RhoA-ROCK Signaling

It has been reported that inhibition of Abl in epithelial cells led to elevated levels of GTP-bound (active) RhoA and enhanced signaling through a direct RhoA effector, the ser/thr kinase ROCK; upregulation of RhoA/ROCK signaling in turn disrupted cadherin-mediated adhesion at adherens junctions in these cells.²⁶ To determine whether Abl functions similarly in myoblasts, we measured levels of GTP-bound RhoA in control C2C12 cells or C2C12 cells depleted of Abl by RNAi. Indeed, Abl-depleted cells showed elevated RhoA activity (Fig. 3A). To assess whether the RhoA-ROCK pathway is involved, we examined whether the phenotype associated with loss of Abl could be reversed by inhibition of ROCK. Treatment with the selective ROCK inhibitor Y27632 partially restored the ability of Abl-depleted C2C12 cells to activate p38α/β and ERK upon N-cadherin ligation (Fig. 3B). Furthermore, Abl-depleted C2C12 cells treated with Y27632 formed aggregates with similar efficiency to control cells (Fig. 2B–E).

Figure 3.

Abl regulates Rho activity in C2C12 cells. (A) Quantification by G-LISA (Cytoskeleton, Inc.,) of GTP-bound RhoA in C2C12 cells stably expressing siRNA against Abl or a control siRNA. Cells were serum starved for 24 hr prior to analysis. *p < 0.01 by Student's t-test. (B) Western blot analysis of pp38, total p38α/β, pERK and total ERK2 in C2C12 cells stably expressing siRNA against Abl, cultured with or without 10 µM Y27632.

Thus, loss of Abl results in activation of RhoA and its downstream effector ROCK to levels that result in inefficient cadherin-mediated C2C12 myoblast adhesion. This offers an explanation for the observation that Abl-depleted cells fail to activate both p38α/β and ERK when plated on N-cadherin substrate. It appears that a pool of Abl protein distinct from that associated directly with Cdo functions to maintain RhoA activity at levels that permit stable cadherin-mediated intercellular adhesions to form, as loss of Cdo only affects N-cadherin-dependent p38α/β activation, not cell aggregation or ERK activation. Taken together, it is concluded that Abl regulates cadherin-mediated p38α/β activation by multiple mechanisms, more generally through regulation of cell-cell adhesion and specifically as a component of Cdo-containing complexes. Interestingly, several studies have reported that RhoA-ROCK signaling must be downregulated during myoblast differentiation in order for fusion into myotubes to occur efficiently.^28–30 It seems likely that one reason this may be so is to allow stable cadherin-dependent adhesive structures to be maintained and that Abl is involved in this process.

Perspective: Regulation of Signaling by Cadherins and their cis-Interacting Partners

N-cadherin ligation initiates binding of Bnip-2/Cdc42 and JLP/p38α/β complexes to the intracellular region of Cdo that is bound in a cis manner to N-cadherin and thereby links extracellular cell-cell contact to activation of a pathway (p38α/β) that controls a cell-type specific transcriptional program.²² Therefore, Cdo acts as a non-ligand-binding signaling coreceptor for N-cadherin in this context. As cadherins are also regulators of cytosketal dynamics and cell shape, such a linkage is an efficient way to couple changes in cell morphology and gene expression during cell differentiation. N-cadherin and other classical cadherins interact with additional transmembrane proteins in a cis manner, although we are not aware of any reported to function similarly to Cdo. For example, fibroblast growth factor receptor-1 (FGFR-1) and N-cadherin associate through their extracellular domains to enhance FGF signaling, but this occurs via N-cadherin's preventing FGFR1 from undergoing ligand-induced internalization.³¹ Nectin-2, an Ig superfamily cell adhesion molecule and N-cadherin interact via their extracellular domains, and they function in concert to ensure proper neural tube formation in Xenopus embryos; however, this effect is independent of Nectin-2's intracellular region.³² The endothelial cell-specific classical cadherin VE-cadherin binds in cis to vascular endothelial protein tyrosine phosphatase (VE-PTP), a receptor-type phosphatase. VE-PTP and VE-cadherin associate through their respective ectodomains, with VE-PTP regulating VE-cadherin phosphorylation state and VE-cadherin-dependent adhesion and cell layer permeability.³³

In addition to a cis association with N-cadherin, Cdo also binds directly to the secreted morphogen, Sonic Hedgehog (Shh) to promote Shh signaling. However, unlike the Cdo-cadherin interaction, Shh-Cdo binding fails to stably attract JLP and Bnip-2 to Cdo and Shh does not activate p38α/β in myoblasts.²² Cdo also binds in a cis manner with the netrin and RGM receptor neogenin, and both neogenin and Cdo are required for the ability of netrin-2 to activate FAK and ERK signaling in myoblasts, although it is not yet clear how Cdo functions in these complexes.³⁴ Taken together, these results indicate that Cdo serves as a multifunctional co-receptor with distinct roles in multiple signaling pathways. The concept of a shared co-receptor offers the potential for cross-regulation of signaling by the pathways involved.²² For example, it is possible that, during development, cells capable of responding to both cadherin ligation and Shh may be able to control signaling strength through these pathways by regulating the ability of Cdo to participate in specific receptor complexes at a given time. This might occur through post-translational modification of the various proteins involved or targeting them to specific subcellular compartments. As the other cadherin-interacting proteins mentioned above also associate with additional transmembrane proteins, it is conceivable that some of the interactions described above may also work in this fashion.